A Massively Parallel Pipeline to Clone DNA Variants and Examine Molecular Phenotypes of Human Disease Mutations

• DOI: 10.1371/journal.pgen.1004819
• Publication Date: 2014-12-11T19:02:22Z
• Authors: Xiaomu Wei; Jishnu Das; Robert Fragoza; Jin Liang; Francisco M. Bast...; Hao Ran Lee; Xiujuan Wang; Matthew Mort; Peter D. Stenson; David N. Cooper; Steven M. Lipkin; Marcus B. Smolka; Haiyuan Yu
• Keywords: Interactome; Massive parallel sequencing; Sanger sequencing

Understanding the functional relevance of DNA variants is essential for all exome and genome sequencing projects. However, current mutagenesis cloning protocols require Sanger sequencing, thus are prohibitively costly labor-intensive. We describe a massively-parallel site-directed approach, "Clone-seq", leveraging next-generation to rapidly cost-effectively generate large number mutant alleles. Using Clone-seq, we further develop comparative interactome-scanning pipeline integrating high-throughput GFP, yeast two-hybrid (Y2H), mass spectrometry assays systematically evaluate impact mutations on protein stability interactions. use this show that disease protein-protein interaction interfaces significantly more likely than those away from disrupt corresponding also find mutation pairs with similar molecular phenotypes in terms both interactions cause same different phenotypes, validating vivo biological our GFP Y2H assays, indicating can be used determine candidate future. The general scheme experimental readily expanded other types interactome-mapping methods comprehensively variants, including non-coding regions.